Interference of components of pegylation reaction mixture in colorimetric protein assays

Suitability of protein quantification method for intended application largely depends on tolerance due to interfering substances in the assay. PEGylation is commonly used procedure to enhance the bioavailability of biotherapeutic proteins. In a PEGylation reaction methoxy-polyethylene glycol aldehyde (mPEG-ALD) is conjugated to protein of interest in presence of cyanoborohydride (CBH) and conjugation reaction is stopped by addition of glycine. Therefore, in the present study components of PEGylation reaction mixture namely mPEG-ALD, CBH and glycine were tested for interference in Lowry, Bradford and Bicinchoninic acid (BCA) assays. The differences in sensitivity of these assays were observed for interfering substances. The mPEG-ALD and glycine interfered in Lowry and BCA assays whereas CBH interfered in all the three assays. Overall Bradford assay was superior to Lowry and BCA assays in quantification of proteins in presence of mPEG-ALD and glycine.